Multistage electromagnetic separator for purifying cells, chemicals and protein structures

ABSTRACT

A multistage electromagnetic separator is designed to separate magnetically susceptible materials suspended in fluids. The apparatus includes an upper plate and a lower plate set to a fill position and the fluid samples are filled into upper and lower cuvettes. The upper cuvette rotates into position above the lower cuvette aligning the upper and lower cuvettes. A translating electromagnet energizes to a programmed current level and translates from the bottom of the lower cuvette to the interface of the plates. The translating electromagnet is de-energized, and a holding electromagnet is energized to a programmed current level pulling particles within a specified mobility range into the top of the captured upper collection cuvette. The holding electromagnet is de-energized leaving the permanent holding magnet to keep the collected sample particles in the top cuvette while the upper plate rotates thereby capturing the sample of the collected particles. The process can be preprogrammed to vary or remain the same for a plurality of capture cuvettes. 
     The apparatus and method of use provides a method for quantitatively separating cells, proteins, or other particles, using multistage, magnetically, electromagnetically assisted separation technology.

This application claims priority from U.S. Provisional application Ser.No. 60,128,627 filed on Apr. 9, 1999 and incorporated herein byreference.

This application is part of a government project, Contract No.NAS9-97027.

TECHNICAL FIELD

1. Field of the Invention

This invention relates an innovative method for quantitativelyseparating cells, chemicals, proteins, and other ligands, or otherparticles, using multistage, magnetically assisted separationtechnology, (“MAGSEP”). MAGSEP is extremely well suited to immunologicalresearch and analysis, pharmaceutical delivery, research and processingand other biomedical applications. Cell separation problems associatedwith clinical, animal, and plant research can be address using MAGSEPtechnology.

2. Description of the Prior Art

Almost all prior art in this field can be classified as magneticfiltration, that is, non-magnetic particles are separated from magneticparticles irrespective of their degree of magnetization. For example,Miltenyi et al., teaches that cells labeled with magnetic particles(paramagnetic, superparamagnetic or ferromagnetic) are trapped in astatic tube or a flowing channel by a strong magnetic field gradientthat causes them to be attracted to said tube or channel wall.Non-magnetic particles are sedimented or convected away, leavingmagnetic particles captive until released from the field and collectedat a later time. In U.S. Pat. No. 5,053,344, Zborowsky applies the term“magnetapheresis”—magnetic stopping, to a similar process. Liberti etal., in U.S. Pat. No. 4,795,698 teach that thin ferromagnetic polepieces extending into a suspension of magnetic particles will attractthem, and only the magnetic particles, to said pole pieces; non-magneticparticles are convected or sedimented away, the field is switched offreleasing the trapped particles into suspension where they are collectedas purified cells. In a chromatography-like approach, Ugelstad teachesthat high field gradients can be established around beaded ferromagneticmedia and fibres, thereby trapping cells labeled with magneticparticles. Other embodiments of these magnetic filtration devices havebeen patented previously as set forth in U.S. Pat. Nos. 4,795,698 and5,053,344. All of these teach a similar, simple binary separation ofmagnetic from non-magnetic particles, and they utilize high-gradientmagnetic fields.

Prior art that is closer to the field of the invention has beenpresented by Powers et al., who teach that a low-gradient magnetic fieldapplied to a horizontally flowing suspension in a channel can trapmagnetically labeled cells dynamically and hence potentially accordingto their level of magnetization by the adsorption of magnetic particles.This method has only been applied to binary separations, however.Winoto-Morbach et al. introduced the concept of “magnetophoreticmobility” implying an intrinsic parameter whereby particles could beseparated according to their speed of migration in a magnetic fieldgradient. Mobility is the ratio of the velocity to the driving force. Inan embodiment that exploits this concept, Zborowsky et al. in U.S. Pat.No. 5,968,820, measured magnetophoretic mobilities and in U.S. Pat. No.5,974,901 teaches that a controlled laminar flow of a suspension ofparticles between large permanent magnet pole pieces results in thedeflection of particles according to their magnetophoretic mobility.Said deflection can be exploited as a means of recovering particlesaccording to their mobilities, or degree of magnetization. Reddy, et.al. (1995) and Zborowski, et al. (1995) have developed analyticalmethods for directly evaluating the magnetization of different magneticparticle types.

Competing alternative preparativetechnologies consist of different typesof separation processes, including electrophoresis and centrifugation.Electrophoresis involves separating materials by passing them through anelectric field with separation occurring based on the attractions of thecells to one particular charge, whether positive or negative. Many ofthe manufacturers in this market are dedicated solely to themanufacturing of electrophoresis equipment. A centrifuge separates cellsand other materials by inertial force. Heavier material is forcedoutward while lighter material remains on the top of the solution. Thisprocess may be beneficial when the cells separated can handle that kindof force and are able to separate based solely on size and/or density.This technique can be especially damaging to a cell, due to the highforces imposed when the unit propels cells into a container wall.

In U.S. Pat. No. 5,974,901, Zborowski et al. teach a method in which anearly constant force field, e.g. magnetic, is applied in a region thatcontains cells that are caused to migrate in the force field. Bycapturing a series of microscope images in the force field, particle(cell) velocities can be measured and, through software, a histogram ofvelocities that indicate the degree of magnetization of the particlescan be produced when the force field is a magnetic force field. Oneapplication of this method is the measurement of magnetophoreticmobility, the ratio of particle velocity to the applied force field,from which additional physical and chemical information about theparticle can be derived. The present invention is distinguished from theZborowski et al reference in that while Zborowski analyzes particles onthe basis of a distribution of magnetic properties, the instantinvention provides a means to capture them on the basis of saidproperties, collecting and separating particles on the basis of theirmagnetophoretic mobility and is not limited to the collection of merelyanalytical data as taught by the Zborowski reference.

In U.S. Pat. No. 5,968,820, Zborowski et al. teach a method in which amixture of biological cells upon whose surface is affixed a number ofmagnetic particles in proportion to the number of receptors of interestto the researcher can be separated on that basis in a flowing stream inwhich they are suspended. The flowing stream flows between two magnetpole pieces, and cells within said stream are deflected toward the polepieces at a velocity that depends on their magnetophoretic mobility andhence magnetic susceptibility and hence receptor density. The separatedcells or particles are finally collected utilizing multiple outlets infractions with each fraction containing cells having a specified rangeof receptor densities. Contrary to the teachings of Zborowski et al.,the instant invention uses a static feed sample in a cuvette and,through the application of magnetic force, causes cells or particles toemerge from said feed cuvette with a velocity that is proportional tomagnetophoretic mobility and hence magnetic susceptibility and hencereceptor density.

In U.S. Pat. No. 5,053,344, Zborowski et al. teaches a system consistingof a gap between two magnetic pole pieces in which a suspension ofparticles is caused to flow through a thin chamber with parallel wallsby gravity or some other driving means,.The chamber is positioned so asto allow the particles suspended in the flowing stream to experience aspatially graded magnetic force. The spatially graded magnetic forcecauses the capture of particles spatially distributed on a planeaccording to their magnetic susceptibility in a process traditionallytermed “ferrography”. Subsequent to capture, some particles, especiallybiological cells, can be examined according to the position at whichthey were captured and classified, but not collected in suspensionaccording to magnetic susceptibility and hence, if labeled with ligandedmagnetic particles, receptor density. This system does not separateparticles collectible in suspension and therein differs from the instantinvention, which is designed to accomplish such separation andcollection.

Improved techniques for separating living cells and proteins areincreasingly important to biotechnology because separation is frequentlythe limiting factor for many biological processes. In response to thatneed, the present invention was developed to provide a method forquantitatively separating cells, particles, ligands, proteins, and otherchemcial species using a magnetic and/or an electromagnetically-assistedseparation process.

SUMMARY OF THE INVENTION

The instant apparatus and method of use provides an innovative methodfor quantitatively separating cells, proteins, or other particles, usingmultistage, magnetically and/or electromagnetically assisted separationtechnology (“MAGSEP”). The MAGSEP technology provides a separationtechnology applicable to medical, chemical, cell biology, andbiotechnology processes. Moreover, the instant invention relates to amethod for separating and isolating mixtures of combinatoriallysynthesized molecules such that a variety of products are prepared, ingroups, possessing diversity in size, length, (molecular weight), andstructural elements. These are then analyzed for the ability to bindspecifically to an antibody, receptor, or other ligate. Such acollection may provide a ligand library containing specific ligands forany ligate even though there are a greater number of conformationsavailable to any one sequence. This technology provides a cellbiologists a tool for studying molecular recognition. Combinationalchemical libraries containing known and random sequences can be surveyedfor strong ligands. Such a tool provides a means of recognizing andisolating agonists, antagonists, enzyme inhibitors, virus blockers,antigens, and other pharmaceuticals.

In clinical applications utilizing a single or multistage magneticand/or electromagnetic separator, cells that are labeled with decreasingnumbers of paramagnetic beads are separated quantitatively on the basisof the extent of labeling by using magnetic fields of increasingstrength. Cells with greater numbers of magnetic beads attached to theirreceptors will be attracted to a weak magnetic field, while cells withfewer beads will not as shown best in FIG. 1. This principle establishesthe basis for separating (“classifying”) cells or other particlesaccording to their magnetic strength, using either a rate or anequilibrium process.

One main reason that electromagnetic field-assisted methods have notbeen heavily employed commercially in the past is the mystique ofequipment used in the field. The physics is considered too complex, butit is rather simple in fact. There is further misunderstanding about themechanism of separation. In addition to the existence of a mystique,real physical factors also have been a deterrent to magneticfield-assisted separations. Most magnetically assisted separations thatrequire the specific adsorption of beaded media to the separand alsorequire some kind of flowing device for removing unwanted particles.

The multistage electromagnetic separator of the instant inventionovercomes these barriers by greatly simplifying the electromagneticfield-assisted separation process. The separator does not require astabilized matrix such as gel, paper, or density gradient. Thetechnology does not require any forced flow of fluid for magneticseparation. The iterative transfer of fluids minimizes flows andprovides a milder and more suitable environment for separating andpurifying cells and proteins. The electromagnetic separator technologyincorporated into the present invention also offers automatic decantingof contaminant suspensions. The unwanted cells or particles are simplyleft behind as by-products of the process in an opposing half chamber.Finally, the end-user of the apparatus will appreciate the addedefficiency of needing to make only one buffer to complete extraction andto collect automatically separated fractions without the complicationsof pumping and volume measurements.

Another application of magnetic separation technology that is in itsinfancy is the development of neoglycoconjugates. Many cells, enzymes,and lectins possess recognition sites for specific carbohydrates(“lectin” means “carbohydrate binding protein”) By conjugating specificcarbohydrates (oligo- or polysaccharides) to the surface of magneticbeads, specific cells, enzymes or lectins can be isolated by HMGS orMACS. This represents an ideal application for MAGSEP, since differentglycoconjugates can be linked to magnetic beads of different strengths,thus separating, out of a mixed population, cells that recognizeglycoconjugate A on strongly magnetizable beads from those thatrecognize glycoconjugate B on weakly magnetizable beads. Furthermore,MAGSEP could also cause the collection of bead-free cells at the end ofthe separation by adding a solution of free sugars that competed for themagnetic binding sitesthreby setting the magnetically captured cellsfree.

In addition to the above very recent innovation, needs for theseparation of cells on the basis of receptor density have beenidentified. Research laboratories have recently used receptor number asa dependent variable in a variety of scientific applications. Inendocrinology mouse leukemia cells exhibit reduced beta-adrenergicreceptors, in growth regulation the number of EGF receptors is regulatedby cell density in cultures which can be modulated by protamine, invirology the cell surface has limited numbers of receptors for herpesvirus glycoprotein D which is required for virus entry into cells, incarcinogenesis the H-ras oncogene alters the number and type of EGF-betareceptors, in infectious diseases galanin receptor levels are coupled topertussis toxin resistance of pancreatic cells, and a diphtheria toxinreceptor-associated protein has been identified. In neurology regulationof opioid kappa receptors occurs in stimulated brain cell cultures, innutrition mast cells lose IgE receptors in protein malnutrition, andvasoactive intestinal peptide (VIP) receptors have been discovered athigh density. This relatively small sample of recent findings indicatesclearly that tools for studying cells with modified receptor densitieswould be welcome.

Methods exist for utilizing high-magnetic-gradient technology for thespecific removal of cells from the human circulation by labeling themwith immunobead ligands. This is now practiced as a binary separationwhich might benefit from continuous separation afforded by the instantinvention.

The use of magnetically delivered therapeutics is another potentialapplication for magnetic particle separation technology.

Once magnetized particles or microcapsules for delivery have been made,it is necessary to separate weakly magnetized particles from those withthe highest susceptibility. Since strongly magnetized particles will berequired, an important consideration is the distance between theexternal magnet and the delivery site and the undesirability ofdelivering weak particles, loaded with drug, to normal-tissue sites toproduce unwanted side effects. The technology may be utilized as a meansfor the separation of a specified subset of T-lymphocytes fortransfusion of AIDS patients, or a specified subset of islet cells forthe treatment of diabetes.

The counting of prepurified cells in diagnostic tests parallelsdevelopments in flow cytometry which costs up to 100 times as much. Thelow cost of this technology can not be overstated: AIDS care givers inthe developing world are puzzled over how to do diagnostic tests thatinvolve flow cytometry in environments that lack flow cytometers. Theinstant invention utilizing a multistage electromagnetic separatorsolves these problems and promises to offer solutions to such globalhealth problems.

In theory, there are no capacity limits to magnetically-assistedseparation. It can be small, for diagnostic purposes, or large, forpreparative applications such as cell transplants. The latter issignificant since a tall magnetic column, which would be required(possibly up to 1 meter and a field greater than 1-2 Teslas) for thequantitative resolution we propose, is replaced by the staged separationcavities in a rotating disk with several modest permanent magnets andelectromagnets as illustrated in FIG. 2.

The development of user-friendly devices that are capable of separatingparticles according to quantity of ligand on their surfaces appears tobe the greatest need in improving magnetically-assisted separationdevices. The magnetic separation industry has made considerable progressin this regard, but the technology to date has been limited to binaryseparation methods. An example would be Baxter Healthcare's Isolex-300Magnetic Cell Separator, which chooses stem/progenitor cells through useof monoclonal antibody (MAB)-coated magnetic beads. The stem cells areselected for reconstituting bone marrow damaged by chemical or radiationtreatment. The instant MAGSEP invention represents a quantum leap inprogress by finally providing a reliable method for differentialseparation on the basis of small differences in surface composition.

Most ligand-based (such as receptor-antibody) cell separation methodsare binary—all or nothing. By combining magnetic attraction, used as arate process, with countercurrent extraction, it is now possible to usemagnetic separation of cells as a quantitative technique, separating onthe basis of the number of ligands bound per cell. This could bequalitative, based on the amount of ligand bound to each kind of cell,or quantitative, based on the amount of ligand bound to cells of thesame type, some with high receptor content and some with low.

It is an object of the present invention to provide a method forquantitatively separating cells, proteins, or other particles, usingmultistage, magnetically, electromagnetically assisted separationtechnology, (“MAGSEP”).

It is an object of the instant invention to provide a method forseparating and isolating mixtures of combinatorial synthesized moleculessuch that a variety of products are prepared, in groups, possessingdiversity in size, length, (molecular weight), and structural elementswhich may be analyzed for the ability to bind specifically to anantibody, receptor, or other ligate, providing a means for forming aligand library containing specific ligands for any ligate to provide acell biologists a tool for studying molecular recognition.

It is an object of the present invention to provide a means ofrecognizing and isolating agonists, antagonists, enzyme inhibitors,virus blockers, antigens, and other pharmaceuticals using combinationalchemical libraries containing known and random sequences.

It is a further object of the present invention to provide a method ofmagnetic cell and cell components sorting for plants and animals.

It is another object of the present invention to develop a plateassembly capable of incorporating at least one and preferably a multipleof magnets, electromagnetic devices, and/or combinations thereof andbase support.

It is another object of the present invention to design electromagnetichardware and drive boards capable of providing variable field strength(in the 1-1000 mT range).

It is another object of the present invention to design an indexingsystem for plate translation.

It is another object of the present invention to incorporate andconfigure the electromagnetic separator of the present invention to fitwithin an ADSEP containment enclosure for space flight and remoteapplications.

It is another object of the present invention to incorporate datamanagement and processing control system.

It is another object of the present invention to provide anelectromagnet exhibiting a relatively quick change in polarity toenhance mixing.

It is another object of the present invention to provide anelectromagnetic separator having a constant force and a formed fluxdensity.

It is an object of the present invention to provide an embodiment,whereby biological cells that have on their surfaces receptors that canbe bound by an antibody can be attached to magnetic particles throughspecific chemical ligands such as avidin, a protein that reacts withbiotin, a vitamin that can be chemically bound to the antibody therebyattaching the cells to magnetic particles to be collected by the presentinvention.

It is another object of the present invention to select homogeneouspopulations of magnetic particles from heterogeneous magnetic particlepopulations synthesized for use in cell research applications.

It is another object of the present invention to select strong,homogeneous populations of magnetic particles for targeted drug deliverywhereby magnetic microparticles are used for the parenteral delivery oftargeted drugs based wherein the differentiation and selection due tothe fact that magnetically weak particles are inimical to this modality.

It is another object of the present invention to utilize an embodimentwherein the translating magnet is a permanent dipole, a permanentquadrupole, or a permanent hexapole magnet, or the magnet is a dipolar,quadrupolar or circular electromagnet.

It is another object of the present invention to utilize an embodimentwherein the translating magnet is a series of fixed electromagnets ofany polarity, operated in sequence so as to sweep particles into acommon starting band.

It is another object of the present invention to utilize an embodimentwherein the control of the translating magnet(s) holding magnet(s) anddisk transfer system is controlled by a computer and custom software.

It is another object of the present invention to utilize an embodimentwherein capture cavities and holding magnets are arrayed in a straightline or some other geometrical relationship especially including in acircle.

It is another object of the present invention to utilize an embodimentwherein more than one sample cuvette, with their translating magnets,serve the array of capture cavities.

It is another object of the present invention to utilize an embodimentwherein the invention is used to separate magnetically labeledbiological cells.

It is another object of the present invention to utilize an embodimentwherein the invention is used to select homogeneous populations ofmagnetic microparticles for application to cell separation and otherbiochemical separation processes.

It is another object of the present invention to utilize an embodimentwherein the invention is used to select homogeneous subpopulations ofmagnetic particles for targeted drug delivery.

It is another object of the present invention to utilize an embodimentwherein the invention is used in any process in which the desired goalis the classification (separation) of magnetic particles according tomagnetophoretic mobility and hence volumetric differentialsusceptibility.

It is another object of the present invention to utilize an embodimentwherein no translation magnet is used.

These and other objects of the present invention will be more fullyunderstood from the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be had uponreference to the following description in conjunction with theaccompanying drawings in which like numerals refer to like partsthroughout the several views and wherein:

FIG. 1 is a magnetic bead attached to a cell receptor by a ligatedspecific antibody;

FIG. 2 is a schematic representation of a multistage electromagneticseparator showing comparison with a hypothetical magnetic chromatographycolumn;

FIG. 3 is a diagram showing a single stage of the magnetic separationprocess wherein cells that bind magnetic beads are drawn along thegradient toward the pole;

FIG. 4 is a partial cutaway view of an electromagnetic separator forsample capture showing the translating and holding magnets andassociated apparatus;

FIG. 5 is an perspective view of an electromagnet separating laboratoryunit showing the plate assembly, the electromagnet assembly, the holdingmagnet, and base unit;

FIG. 6 is an embodiment of a translating electromagnet showing a steelcore and windings;

FIG. 7 shows the plate assembly used in the embodiment of FIG. 5;

FIG. 8 is a perspective view showing the plate assembly fill ports ofthe embodiment of FIG. 5;

FIG. 9 is a cuvette utilized in the embodiment of FIG. 4 further showinga capture cuvette and sample cuvette together with the holdingelectromagnet, permanent holding magnet, and translating electromagnet;

FIG. 10 is a cross-sectional view of the plate and a cuvette showingfiling of the sample cuvette;

FIG. 11 is a partial cutaway view of the plate and a cuvette showing theposition of the cuvette with respect to the rotation of the top plate;

FIG. 12 is a partial cutaway view of the plate and a cuvette showinginitiation of particle alignment in a sample cuvette due to thetranslation magnet energizing and moving particles toward the plateinterface;

FIG. 13 is a partial cutaway view of the plate and a cuvette showingposition of the translation magnet and capture of particles;

FIG. 14 is a partial cutaway view of the plate and a cuvette showingrotation of the top plate to capture a fraction of particles;

FIG. 15 is a graph showing the translating magnet field strength;

FIG. 16 shows the holding magnet assembly of the embodiment of FIG. 4;

FIG. 17 shows a graph depicting the separation of magnetic fromnon-magnetic micro spheres;

FIG. 18 is an exploded perspective view showing a plate assembly forattachment to a translating electromagnetic station;

FIG. 19 is an exploded perspective view showing an indexing system forMAGSEP for rotating the collection plate;

FIG. 20 is a perspective view showing a modular design of the processingunit providing a cassette change out;

FIG. 21 is a perspective view showing a MAGSEP cassette occupying thesame form factor as the space flight proven ADSEP cassette providingchange out capabilities;

FIG. 22 is an alternate embodiment showing a translating magnet assemblyutilizing multiple quadropole magnets energized sequentially in acascading magnet design;

FIG. 23 is a an alternate embodiment showing a translating magnetassembly consisting of a moving quadruple magnet; and

FIG. 24 is an alternate embodiment showing a quadruple or hexapoletranslating magnet.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is an electromagnet separator 10 forquantitatively separating substrates including cells, proteins, ligands,chemicals, antigens, and other particles by using an electromagneticallyassisted separation process. The multi-stage electromagnet, (“MAGSEP”),10 of the present invention allows a multiple stage separation based onmagnetic susceptility and magnetophoretic mobility. The preferredembodiment of the electromagnet separator 10 is a multistagecounter-current device in which the substrates or cells are labeled withdecreasing numbers of paramagnetic beads and separated quantitatively onthe basis of the extent of labeling by using magnetic fields ofincreasing strength. The electromagnetic separator 10 enhances productrecovery by collecting fractions automatically and provides differentialseparation where only binary separations were previously possible. Itwill work with any aqueous suspension and has the flexibility to operateefficiently in commercial applications and space research laboratories.The invention makes it possible to separate large quantities ofimmunological, hematological, and other differentiating cell types indirect proportion to their surface antigen content. Moreover, it makesit possible to either refine samples to a higher level or purity ofcategorize portions of the sample based or magnetic susceptibilityand/or magnetophoretic mobility. Moreover, the field strength can bevaried to produce uniform capture of magnetized cells or othersubstrates.

Magnetophoretic mobility is defined as:$\mu_{m} = \frac{v_{m}}{B - \frac{B}{Z}}$

where B is the capture magnet's magnetic field strength and v_(m) is thevelocity of the particle in the magnetic field. The velocity is afunction of the magnetic field and properties of the particle and thesolvent:$v_{m} = \frac{2a^{2}\Delta \quad {xB}{B}}{a\quad {\eta\mu}_{o}{Z}}$

Therefore, each stage in the MAGSEP device selects particles ofdifferent magnetophoretic mobilities. The particles in each of thestages will have a different mobility distribution. The low magneticfield strengths will select particles of higher mobility, whereas thehigher magnetic field strengths will select for lower mobilities.Therefore, each stage will contain a magnetophoretic mobility cutoff,based on the magnetic field strength of the capture magnet, and thedwell time of the capture.

In equation (2) a is a particle radius, ΔX is the magneticsusceptibility difference between particle and medium, η is viscosity,and μ is the magnetic permeability of free space.

The method of cell separation using a magnetic field has beenimplemented as a binary separation between cells that have and have notbound magnetic micro spheres on the basis of a specific surface ligand,as best shown in FIG. 1. As shown an antigen is attached to a cellreceptor site and biotin is attached to the antibody. A magnetic bead isattached to avidin which is connected to the biotin. Since biologicalcells that have on their surfaces receptors that can be bound by anantibody can be attached to magnetic particles through specific chemicalligands such as avidin, a protein that reacts with biotin, a ligand canbe chemically bound to the antibody.

FIG. 2 is a schematic representation of a multistage electromagneticseparator showing comparison with a hypothetical magnetic chromatographycolumn. As noted heretofore, the MAGSEP device utilizes a step-wiserotary distribution and containment system which selects, isolates, andstores particles of different magnetophoretic mobilities. The particlesin each of the stages will have a different mobility distribution. Thelow magnetic field strengths will select particles of higher mobility,whereas the higher magnetic field strengths will select for lowermobilities. Therefore, each stage will contain a magnetophoreticmobility cutoff, based on the magnetic field strength of the capturemagnet, and the dwell time of the capture. FIG. 2 demonstrates that thefast cells have the greater magnetophoretic mobility. Thus, the cellsare separated according to the quantity of ligand on their surfaces.

By combining magnetic attraction, used as a rate process, withcountercurrent extraction, it is possible to use magnetic separation ofcells as a quantitative technique separating on the basis of the numberof ligands bound per cell. This could be qualitative, based on theamount of ligand bound to each kind of cell, or quantitative, based onthe amount of ligand bound to cells of the same type, some with highreceptor content, and some with low receptor content.

FIG. 3 is a diagram showing a single stage of the magnetic separationprocess whereby cells that bind magnetic beads are drawn along thegradient toward the pole. The illustration shows a magnetic source,either permanent or electromagnetic, at the top of the container orcuvette, which produces a magnetic field gradient therein. Magneticforce creates movement of the paramagnetic particles in accordance withtheir magnetophoretic mobility. The electromagnetic separation device 10of the present invention provides a very clean separation wherein theparticles are loosely aligned in strata with the most magnetic particlesat the top of the cuvette, particles with a lower magnetic suceptiblityare suspended in the middle, and particles with little or no magneticsusceptibility are suspended in the bottom of the cuvette.

For example, all separands attached to magnetized particles such ascells or proteins may be drawn into a half-cavity of a multistageseparator from a uniform suspension, while non-magnetic separands remaindistributed equally between upper and lower cavities. Nonmagneticparticles are allowed to settle for a predetermined time period. Theupper cavity is moved to a position above a fresh solution that isthoroughly mixed with the separated cells. In low gravity, the resultmay be achieved not by sedimentation, but by dilution of non-magneticcells out of the cavity.

The preferred embodiment achieves multi-stage separation by utilizingmultiple sample cavities within the same plate assembly. The fieldstrengths of both the translating electromagnet and the holdingelectromagnet can also be varied during the separation process.

FIG. 4 is a perspective view of an embodiment of a multistageelectromagnetic separator 10 of the present invention. The MAGSEP unit10 illustrates the upper plate 26 rotatively cooperatively engaging alower plate 24 supported by a plurality of leg members 22 whereby theupper plate 26 contains at least one and preferably a plurality of uppercollection cuvettes 27 in selected fluid communication with the lowerplate 24 and a lower sample cuvette 38 disposed therein wherein a sealis formed thereinbetween with a sealant such as a grease, wax, or otherlubricating and/or sealing constituent.

FIG. 4 also shows a translating electromagnet 40, a translation system42, a holding magnet 44 which is a permanent magnet in the embodiment, aholding electromagnet with cooling fan 46, a plate rotation system 48,and a plate location microswitches 50.

As illustrated in FIG. 5, a commercial unit is shown wherein the upperplate 26 is formed of a polymer such as a polycarbonate and is mountedonto a bearing 33 and secured with a clamping bolt 29. The legs support22 are replaced by flanges 23 forming a base. The lower plate 24 isformed of stainless steel. A holding magnet stepper motor 31 rotates thetop plate 26. The holding electromagnet 46 is suspended over the uppercuvettes 27. An electromagnet 35 is shown within the base. The base ismounted onto a housing 37 which includes a power switch 39, 110 VAC plug41, communications port 43, indicator lights 45, and cooling fan 47.

More particularly, the laboratory unit includes a computer and software,and consists of an electronics housing and the processing unit. Theelectronics box has several interfaces including 110 VAC, power switch,RS 232 interface, and status lights. The unit receives power through the110 AC connector. Power is activated with the power switch. The PC thatcontrols the unit operates via the RS232 signal connector. The status ofthe power, translating electromagnet, holding magnet, and plate rotationare indicated with a graphical user interface via a personal computer.

A single processing unit consists of the upper and lower plates, platerotation system electromagnet, electromagnet translation system, andholding magnet assembly. The plates bolt together through a taperedroller bearing that allows the plates to rotate with respect to oneanother The lapped interface between the plates provides a sealseparating the fluids. The lower cuvette can be aligned with as many as15 upper cuvette stations during processing. A two-phase stepping motorrotates the upper plate by driving the rotation system that engages aninternal gear mounted to the underside of the upper plate. Thetranslating electromagnet is mounted to the translation system thattranslates the electromagnet vertically along the lower cuvette. Aprogrammed amount of current is sent to the electromagnet creatingmagnetic field across the lower cuvette. The translating electromagnetfield strength can be programmed from 0 to 1400 gauss (measured at thepoleface), or other selected range. The electromagnet translation systemmoves the electromagnet up and down the lower cuvette. The translationrates can be programmed to range from 5 micrometers/second to 2000micrometers/second or other selected values. The holding magnet assemblyconsists of a permanent magnet mounted on an arm that is connect to astepping motor. The stepping motor rotates the arm containing theholding magnet, positioning the holding magnet above the cuvette beingprocessed.

As best shown in FIG. 6, one preferred embodiment of a translatingelectromagnet 40 consists of a C-1018 steel core 42 with 818 windings of26-gage copper magnet wire formed in a disk having an air gap inbetweenthe distal ends thereof. It receives current ranging from 0 to 2.16 Ampsfrom the electronics box. The magnetic field strength can be programmedfrom 0-1500 gauss (measured at the poleface). The electromagnetictranslation system moves the electromagnet up and down the lower cuvette28. The translation rates can be programmed to range from 120 to 250μms.

As best shown in FIG. 4, the holding magnet 44 assembly consists of apermanent magnet mounted on an arm 19 that is connected to a steppingmotor 31. The stepping motor 31 rotates the arm 19 containing theholding magnet 44, positioning the holding magnet 44 above the uppercuvette 27 being processed.

Method of Use

MAGSEP 10 was designed to separate magnetically susceptible materialssuspended in fluids. An application of the embodiment shown in FIG. 4 isas follows:

The upper plate 26 and lower plate 24 are set to the fill position (halfstepped), and the fluid samples are filled into the upper 27 and lowercuvettes 28. The upper cuvette 27 rotates into position above the lowercuvette 28 aligning the upper 27 and lower cuvettes 28. The translatingelectromagnet 40 energizes to a programmed current level and translatesfrom the bottom of the lower cuvette 28 to the interface of the plates24, 26. The translating electromagnet 40 is de-energized, and theholding electromagnet 46 is energized to a programmed current levelpulling particles within a specified mobility range into the top of thecaptured upper collection cuvette 27. Finally, the holding electromagnet46 is de-energized leaving the permanent holding magnet 44 to keep thecollected sample particles in the top cuvette 27 while the upper plate26 rotates thereby capturing the sample of the collected particles. Thisprocess can be preprogrammed to vary or remain the same for up to 15capture cuvettes 27.

FIG. 7 is a cross-section of the plate assembly showing the bottom plate24 in cooperative engagement with the upper plate 26 in alignment with asample cuvette 28 and an upper collection cuvette 27 and the holdingmagnet 44 well of the arm 19.

More particularly, FIG. 8 shows the filling ports within a section of atop plate 26 in fluid communication with the upper collection cuvettes27. Also shown in FIGS. 8 and 10 is a sample fill port 52, sample ventport 53, sample drain port 54 (FIG. 10), and fill relief port 56. Theplate assembly holds the samples before and after separation. The plateassembly of one preferred embodiment consists of a polycarbonate topplate, a stainless steel bottom plate, and one polycarbonate samplecuvette 28. The top plate is bolted to the bottom plate with a centralclamping bolt that serves as an axle and allows the top plate to rotatewith respect to the bottom plate. The top plate has at least one andpreferably a plurality, 15 as shown, of cavities called collectioncuvettes 27. The sample cuvette 28 is attached to an opening in thebottom plate 24. This allows the collection cuvette 27 to be rotatedover the sample cuvette 28, thus allowing particles in the samplecuvette 28 to be transferred to the collection cuvette 27. Thecollection cuvette can then be rotated away from the sample cuvettecapturing the contents of the collection cuvette. The pressure of theclamping bolt seals the top plate to the bottom plate.

FIGS. 9-14 show the step-wise progression of separating particlesutilizing the present invention.

As shown in FIG. 9, the cuvette configuration shows the position of thecapture cuvette 28, sample cuvette 38, holding electromagnet 46,permanent holding magnet 44, and translating electromagnet 40. FIG. 10illustrates filling the sample cuvette 28 with cells or other substratehaving magnetic particles selectively attached thereto. As shown in FIG.11, the top plate 26 rotates with respect to the bottom plate 24 and thesample cuvette 28 to a full step position with sample and collectioncuvettes finally aligned. The translational electromagnet 40 energizesand moves toward the plate interface as depicted in FIG. 12 showinginitiation of particle alignment in the sample cuvette 28. It should benoted that the sequence for filling can be to raise the translationalelectromagnet 40 with the upper plate 26 one-half stepped, then bringthe upper collecting cuvette 27 holding the magnet in place, or to bringthe upper chamber 27 of the cuvette and magnet 40 into place, thenelevate the sample cuvette 28.

FIG. 13 shows the final position of the translating electromagnet andcapture of particles wherein the translating electromagnet 40 stops anddeenergizes, and the holding electromagnet 46 energizes, and fieldcouples with the permanent magnet 44. Finally, as shown in FIG. 14, thetop plate 26 is rotated to capture a selected fraction of the particlesas the process sample.

FIG. 15 is a graph depicting the translating magnet 40 field strength ofan embodiment such as described in FIG. 4.

As shown in FIG. 16, the capture or holding electromagnet 46 orprogrammable electromagnet is used to pull the sample past the plateinterface and into the top of the upper cuvette 27.

The permanent magnet 44 is used to keep the captured sample at the topof the capture cuvette 27, preventing it from falling into the plateinterface and becoming trapped between the plates 24, 26. The permanentmagnet 44 size and materials can be varied to provide a variety of fieldstrengths.

FIG. 17 is a graph showing the results of a separation experimentseparating magnetic from non-magnetic microparticles by the multistagemagnetophoresis process. The experiment began with a mixture containing90% 1-2 μm magnetic spheres (“animospheres, Polysciences, Inc.) and 10%6.0 μm non-magnetic spheres (Interfacial Dynamics Corp.). The particlesmay be suspended in any type of fluid; however, water, polyethyleneglycol, or ethyl alcohol are typically used. Six cavities were equippedwith magnets ranging from 10 mT to 375 mT field at the pole face.Gradients were estimated using field measurements at 2.54 cm andconverted to mT/m. Dwell time at each cavity was 15 min, and traveldistance was on average 3 mm. From these data, a magnetophoreticmobility was estimated for each of the 7 cavities, as given on theaccompanying graph.

It is seen that 80.1% of the magnetic particles were all captured incavity #6, corresponding to a mobility of 0.6 mm/N-s, where only 2.8% ofthe non-magnetic particles were captured. The “purity” of the magneticspheres went from 90% to 99.6%.

FIG. 18 is an exploded perspective view showing an external plateassembly for a translating electromagnetic station, wherein the plateassembly 100 includes a translating electromagnetic station 102(preferably three per sample plate 104) attached to a sample plate 104in rotational fluid communication with a plurality of cavities 106formed and aligned around the periphery of a collection plate 108 whichis in cooperative engagement with a holding magnet (electromagnet) 146.

FIG. 19 is an exploded perspective view showing an indexing system forMAGSEP for rotating the collection plate, wherein a tray cover 110attaches to the plate assembly 100 which is connected to a worm gear 112and providing an angular contact bearing 114 connected to a bearingstandoff 116. The assembly is rotatively attached to a base assembly 119having a bearing race relief 118, and position sensor 120, wherein thebase 119 forms a tray 122 which is mechanical connection with shaft 124of a precision worm 126 in communication with a flexible shaft coupling128 driven be a stepper motor 130. The indexing system tray 58 isdisposed within a cartridge or cassette 132 defined by a containmentenclosure 134 and cover 136 holding the plate assembly as shown in FIG.20 which is a perspective view showing a modular design of theprocessing unit providing a cassette change out.

As shown in FIG. 21, a MAGSEP cassette can be utilized in a modulardesign including a processing module holding more of the same ordifferent cassettes.

As an alternate embodiment, FIG. 22 shows the use of a cascading magnetsystem in which a series of dipole, quadrupole or ring magnets, saythree or four, is stacked along the upper cylindrical cavity of theMAGSEP two-plate device. These are activated in sequence, lowest first,to accelerate (in the sense of a magnetic induction accelerator as usedin particle physics) particles upward until they reach an unstable pointas defined by Earnshaw's theorem, at which time the first field isswitched off and the second switched on to continue the upward captureprocess without sticking the particles to the wall by magnetapheresis asset forth and described in U.S. Pat. No. 5,053,344 by Zborowski et al.,1995, hereby incorporated by reference.

FIG. 2 is this alternate embodiment showing a translating magnetassembly utilizing multiple quadropole magnets energized sequentially ina cascading magnet design consisting of a sample cuvette, separationelectromagnet, collection cuvette, and holding electromagnet.

FIG. 23 is an alternate embodiment showing a translating magnet assemblyconsisting of a moving quadruple magnet consisting of a separationelectromagnet, sample cuvette, collection cuvette, and holdingelectromagnet.

FIG. 24 is an alternate embodiment showing a quadruple or hexapoletranslating magnet.

Alternate Applications

The present invention could also be used as a means of “MagneticChromatography”. Capture can be “isocratic”, wherein magnets in all ofthe stages have equal strength, or “gradient” wherein magnets atincreasing stage numbers have increasing field strength. In the lattercase, in a typical application the first stage would have no magnet andno upper cavity and would serve the purpose of homogenizing the cellmixture by stirring just before the beginning of transfers. The secondstage would have no magnet and would serve the purpose of addingmagnetic particles to the cell suspension from a low volume uppercavity, mixing them together, and allowing them to react. The thirdstage would have a very weak magnet in the upper cavity, which wouldhave similar volume to the lower cavity, and would attract only the mosthighly magnetized cells, namely those with the most receptors for themagnetic ligand. The fourth stage would have a stronger magnet than doesthe third in its upper compartment and would attract more weaklymagnetized cells, etc. until, at the final-but-one stage the strongestmagnet of all would capture the cells with the least receptors. Thefinal stage would also have no magnet and would contain any remainingcompletely unmagnetized cells after the final transfer. In the presenceof gravity uncaptured cells will settle into the lower cavities bygravitational sedimentation if the transfer times are made sufficientlylong. In the absence of gravity uncaptured cells would remain in boththe upper and lower cavities at each transfer; however, continued mixingwith each transfer would have the effect of removing the uncapturedcells in each cavity.

The foregoing detailed description is given primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom, for modification will become obvious to those skilled in theart upon reading this disclosure and may be made upon departing from thespirit of the invention and scope of the appended claims. Accordingly,this invention is not intended to be limited by the specificexemplifications presented herein above. Rather, what is intended to becovered is within the spirit and scope of the appended claims.

We claim:
 1. A method for quantitatively separating cells, proteins orother particles, and collecting at least some of said particles in aband using a multistage electromagnetic separator including a series ofdipole, quadrupole or ring magnets, stacked along an upper cavity of anupper plate in rotational alignment with a lower plate including atleast one sample container containing particles in a fluid in sealedfluid communication and cooperatively engaging said upper plate;comprising the steps of: activating at least one magnet providing aselection of field strengths in sequence, lowest field strength first,forming an electric field to accelerate said particles susceptible tosaid selected field strength upward until they reach an unstable pointas defined by Earnshaw's theorem defining migrating particles, at whichtime a first field is switched off and a second field is switched on tocontinue the upward capture process without sticking said migratingparticles to the wall by magnetophoresis.
 2. A multistageelectromagnetic separator for separating magnetically susceptiblematerials suspended in fluids, comprising: a frame supporting a lowerplate including at least one sample cuvette disposed therein containingat least one magnetic particle suspended in a fluid; an upper plate incooperative engagement with said lower plate, said upper plate includingat least one collection cuvette disposed therein containing fluidalignable with said sample cuvette in said lower plate for fluidcommunication therewith; a translating electromagnet energizable to aprogrammed current level translating from a bottom of said samplecuvette to the interface of said upper plate and said lower plate; aholding electromagnet is energizable to a programmed current level forpulling said at least one magnetic particle within a specified mobilityrange into the top of said at least one collection cuvette; means forrotating said upper plate with respect to said lower plate; and meansfor sealing said at least one magnetic particle within said at least onecollection cuvette.
 3. An electromagnetic separator for separatingparticles suspended in a fluid according to their magnetophoreticmobility, comprising: a base unit including means for controlling, meansfor supporting, and means for providing electrical power to a platerotation apparatus; said plate rotation apparatus including a holdingmagnet, and a plate assembly; said plate assembly comprising a firstplate containing at least one collection cavity in selected fluidcommunication with and cooperatively engaging a second plate includingat least one sample container holding said particles in said fluiddisposed therein, said first plate moving with respect to said secondplate; means for moving said first plate with respect to said secondplate; means for aligning said first plate with respect to said secondplate; a translating magnet for generating an electric field; means forcontrolling the movement of said first plate or said second plate, saidtranslating electromagnet, and said holding magnet.
 4. Theelectromagnetic separator of claim 3, wherein said holding magnet isselected from the group consisting of an electromagnet and a permanentmagnet.
 5. The electromagnetic separator of claim 3, wherein said firstplate contains a plurality of collection cavities.
 6. Theelectromagnetic separator of claim 3, wherein collection cavitiescomprise collection cuvettes.
 7. The electromagnetic separator of claim3, wherein said sample containers are sample cuvettes.
 8. Theelectromagnetic separator of claim 3, wherein including a means forcooling said electromagnet.
 9. The electromagnetic separator of claim 3,including means for rotating said first plate with respect to saidsecond plate.
 10. The electromagnetic separator of claim 3, wherein saidfirst plate is an upper plate.
 11. The electromagnetic separator ofclaim 3, wherein said second plate is a lower plate.
 12. Theelectromagnetic separator of claim 3, including a holding magnet foreach collection cavity.
 13. The electromagnetic separator of claim 3,including an individual translational magnet for each sample.
 14. Theelectromagnetic separator of claim 3, wherein said collection cavitiesand said holding magnets are arrayed in a geometric relationship. 15.The electromagnetic separator of claim 3, wherein said geometricrelationship is linear.
 16. The electromagnetic separator of claim 3,wherein said geometric relationship is circular.
 17. The electromagneticseparator of claim 3, including a plurality of samples serving an arrayof said collection cavities.
 18. The electromagnetic separator of claim3, wherein said translating magnet comprises a series of fixedelectromagnets of a selected polarity, operated in sequence so as tosweep said particles into a common starting band.
 19. Theelectromagnetic separator of claim 3, wherein said sample is moved withrespect to at least one stationary translating electromagnet of aselected polarity.
 20. The electromagnetic separator of claim 3, whereinsaid translating magnet and said holding magnet is controlled by acomputer.
 21. The electromagnetic separator of claim 3, wherein saidtranslating magnet comprises a single permanent or electromagnet forminga common starting band of particles at a selected position with saidsample container.
 22. The electromagnetic separator of claim 3, whereinsaid first plate includes a fill relief and vent port.
 23. Theelectromagnetic separator of claim 3, wherein said first plate includesa sample vent port and fill port.
 24. The electromagnetic separator ofclaim 3, wherein said translating magnet comprises a permanent dipole, apermanent quadrupole, a permanent hexapole magnet, a dipolar magnet, aquadrupolar electromagnet, or a circular electromagnet.
 25. Theelectromagnetic separator of claim 3, wherein said translating magnetcomprises a series of fixed electromagnets of any polarity, operated insequence so as to sweep particles into a common starting band.
 26. Theelectromagnetic separator of claim 3, wherein control of saidtranslating magnet, said holding magnet, and said disk transfer systemis controlled by a computer.
 27. The electromagnetic separator of claim3, wherein said collection cavities and said holding magnets are arrayedin a geometrical relationship of linear array.
 28. The electromagneticseparator of claim 3, wherein said collection cavities and said holdingmagnets are arrayed in a geometrical relationship of a circle.
 29. Theelectromagnetic separator of claim 3, wherein said at least onecollection cavity is aligned using an analytical device.
 30. Theelectromagnetic separator of claim 3, including means selected from thegroup consisting of a wax or grease for forming a seal between saidfirst plate and said second plate.
 31. An electromagnetic separator forseparating particles suspended in a fluid according to theirmagnetophoretic mobility, comprising: a base; a holding magnet; a firstplate containing at least one collection cavity in selected fluidcommunication with and cooperatively engaging a second plate includingat least one sample holding said particles in said fluid disposedtherein, said first plate moving with respect to said second plate;means for moving said first plate with respect to said second plate;means for aligning said first plate with respect to said second plate;means of electrical power; means for controlling the movement of saidfirst plate or said second plate and said holding electromagnet.
 32. Theelectromagnetic separator of claim 31, including a translatingelectromagnetic.
 33. The electromagnetic separator of claim 31, whereinsaid translating electromagnet.
 34. The electromagnetic separator ofclaim 31, including means for sealing said first plate in fluidcommunication with said second plate.
 35. The electromagnetic separatorof claim 31, wherein said means for sealing comprises grease, wax, andlubricant.
 36. The electromagnetic separator of claim 31, wherein aholding magnet stepper motor rotates said first plate.
 37. Theelectromagnetic separator of claim 31, wherein said holding magnet is a15× permanent magnet.
 38. The electromagnetic separator of claim 31,wherein said holding magnet is a permanent magnet mounting on an armconnecting to a stepping motor, said stepping motor rotating said armcontaining said holding magnet positioning said holding magnet withrespect to said cavity.
 39. The electromagnetic separator of claim 31,including an indexing system for rotating said first plate, saidindexing system being disposed withing a cartridge.
 40. Theelectromagnetic separator of claim 31, wherein said translational magnetincludes translation rates range up to 420 micrometers per second. 41.The electromagnetic separator of claim 31, wherein said translationalmagnet includes translation rates range down to 5 micrometers persecond.
 42. The electromagnetic separator of claim 31, wherein saidtranslation magnet has a field strength programmable in a range of from0 to 1400 gauss measured at the poleface.
 43. An electromagneticseparator for separating magnetically susceptible materials suspended influids, comprising: a frame supporting a lower plate including at leastone sample container disposed therein containing at least one magneticparticle suspended in a fluid; an upper plate in cooperative engagementwith said lower plate, said upper plate including at least onecollection cavity disposed therein containing fluid alignable with saidsample container in said lower plate for fluid communication therewith;a translating electromagnet energizable to a programmed current leveltranslating from a selected point of said sample container to a selectedposition below the interface of said upper plate and said lower plate; aholding electromagnet is energizable to a programmed current level forpulling said at least one magnetic particle within a specified mobilityrange into the top of said at least one collection cavity; means forrotating said upper plate with respect to said lower plate; and meansfor sealing said at least one magnetic particle within said at least onecollection cuvette.
 44. A method of collecting a particle sample with aplate assembly, comprising the steps of: cooperatively engaging a firstplate with a second plate, said second plate including at least oneopening therein, said second plate cooperatively engaging said firstplate and forming a seal thereinbetween; disposing at least one particlein at least one sample container, said sample container being in fluidcommunication with said at least one opening; rotating and aligning saidopening in said second plate and said at least one sample container withsaid collection cavity in said first plate; transferring said at leastone particle in said sample container into said collection cuvette; androtating said second plate and said sample container with respect tosaid first plate and said collection cuvette capturing said at least oneparticle within said collection cuvette of said first plate.
 45. Amethod for quantitatively separating particles with a multistageelectromagnetic separator, comprising the steps of: activating insequence a series of magnets comprising at least a first magnet and asecond magnet stacked along a cavity of multiplate separator having atleast a first plate and a second plate in cooperative engagement withone another; creating a first magnetic field with said first magnet anda second magnetic field with said second magnet; accelerating saidparticles until said particles reach an unstable point; switching offsaid first magnet and said first magnetic field; switching on saidsecond magnet and said second magnetic field moving said particles to aselected point without sticking said particles to a wall of said cavity;separating said particles based on its magnetophoretic mobility; andcapturing said particles.
 46. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of achieving multi-stage separation by utilizingmultiple cavities with said plate.
 47. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of varying the field strength of saidtranslating magnet during said separation process.
 48. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of suspending said holdingmagnet over said cavity.
 49. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of varying the field strength of said holding magnetduring said separation process.
 50. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of using a step-wise rotary distributionand containment system for selecting, isolating, and storing particlesof different magnetophoretic mobilities.
 51. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of each stage containing amagnetophoretic mobility cutoff based on the magnetic field strength ofthe collection magnet and the dwell time of the collection.
 52. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said particles comprisecells, proteins, enzymes, chemicals, ligands, lectins, and combinationsthereof.
 53. The method for quantitatively separating particles with amultistage electromagnetic separator of claim 45, wherein said cavity isa cylindrical cavity.
 54. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,wherein said magnet is selected from the group consisting of at leastone dipole magnet, at least one quadruppole magnet, at least onehexapole magnet, at least one circular electromagnet, at least onepermanent dipole magnet, at least one permanent quadrupole magnet, atleast one permanent hexapole magnet, and combinations thereof.
 55. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said first magnetcomprises a series of fixed electromagnets of any polarity, operated insequence so as to sweep particles into a common starting band.
 56. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said translatingelectromagnet moves up and down said sample container.
 57. The methodfor quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said particles arecontained in a sample cuvette.
 58. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, wherein said particles emerge from said sample container at avelocity that is proportional to magnetophoretic mobility and magneticsusceptibility.
 59. The method for quantitatively separating particleswith a multistage electromagnetic separator of claim 45, including thestep of labeling said particles with liganded magnetic particles andseparating said particles by its receptor density.
 60. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, wherein said particles are suspended in asolution.
 61. The method for quantitatively separating particles with amultistage electromagnetic separator of claim 45, including the step ofstratifying in layers said particles within said sample container intofast particles, magnetized particles, slow particles, and sedimentingnon-magnetic particles.
 62. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of removing said layers of said stratified particlesinto individual collection cavities.
 63. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of isolating mixtures of combinatoriallysynthesized molecules into groups of products according to its physicaland/or chemical properties.
 64. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,wherein said chemical and/or physical properties comprises its size,length, shape, molecular weight, and structural elements.
 65. The methodfor quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step of analyzingsaid particles for the ability to bind specifically to an antibody,receptor, or the ligate.
 66. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of collecting said analyzed particles forming aligand library containing a greater number of conformations available toany one sequence for studying molecular recognition.
 67. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of combining ligand librariesforming combinational chemical libraries containing known and randomsequences and surveying said libraries for strong ligands andrecognizing and isolating agonists, antagonists, enzyme inhibitors,virus blockers, antigens, pharmaceuticals, and combinations thereof. 68.The method for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step of separatingsaid particles according to their magnetic strength by rate.
 69. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step of separatingsaid particles according to their magnetic strength by an equilibriumprocess.
 70. The method for quantitatively separating particles with amultistage electromagnetic separator of claim 45, including the step ofdecanting contaminant suspensions of unwanted particles by leaving themin the sample container as by-products of the process.
 71. The methodfor quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said sample is containedin a cuvette.
 72. The method for quantitatively separating particleswith a multistage electromagnetic separator of claim 45, including thestep of making only one buffer for completing collection of saidparticles.
 73. The method for quantitatively separating particles with amultistage electromagnetic separator of claim 45, including the step ofcollecting said particles in separated fractions.
 74. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step selecting particles comprisingcells, enzymes, and lectins having specific carbohydrate recognitionsites, conjugating a specific carbohydrate on a surface of a magneticbead of a selected magnetic strength forming a glycoconjugate, andseparating from a mixed population particles that recognize a firstglycoconjugate on a strongly magnetizable bead from particles thatrecognize a second glycoconjugate on a weakly magnetizable bead.
 75. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step ofdifferential separation n the basis of small differences in surfacecomposition of said particle.
 76. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of using countercurrent extraction toseparate particles quantitatively on the basis of the number of ligandsbound to particles of the same type some with high receptor content andsome with low receptor content.
 77. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of using countercurrent extraction toseparate particles qualitatively based on the amount of ligand bound toeach kind of particle.
 78. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of sorting particles comprising cell components ofplants and animals.
 79. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,wherein said magnetic field has a variable field strength.
 80. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, wherein said magnetic field has avariable field strength of up to 1000 mT.
 81. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of positioning said firstplate with respect to said second plate by an indexing system for platetranslation.
 82. The method for quantitatively separating particles witha multistage electromagnetic separator of claim 45, including the stepof configuring said method for insertion into an ADSEP containmentenclosure for space flight and remote applications.
 83. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of incorporating datamanagement and a processing control system therewith.
 84. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, wherein said first magnet is an electromagnethaving a relatively quick change in polarity to enhance mixing.
 85. Themethod for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step of creating aconstant force and a formed flux density.
 86. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 45, including the step of selecting particlescomprising biological cells that hove on their surfaces receptors thatcan be bound by an antibody attached to magnetic particles through aspecific chemical ligand that can be chemically bound to the antibodythereby attaching said biological cells to magnetic particles forcollection.
 87. The method for quantitatively separating particles witha multistage electromagnetic separator of claim 45, including the stepof selecting particles having homogeneous populations of magneticparticles from heterogeneous magnetic particles synthesized for use incell research applications.
 88. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of selecting strong, homogeneous populations ofmagnetic particles from targeted drug delivery whereby magneticmicroparticles are used for the parenteral delivery of targeted drugsbased wherein the differentiation and selection thereof is due to thefact that magnetically weak particles are inimical to the modality. 89.The method for quantitatively separating particles with a multistageelectromagnetic separator of claim 45, including the step of separatingmagnetically labeled biological cells.
 90. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of labeling biological substrates withdecreasing numbers of paramagnetic beads and separating quantitativelysaid substrates on the basis of the extent of labeling by using magneticfields of increasing strength.
 91. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of labeling biological substrates withdecreasing numbers of paramagnetic beads and separating quantitativelysaid substrates on the basis of the extent of labeling and the dwelltime of the collection.
 92. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of selecting homogeneous populations of magneticmicroparticles for application to cell separation and biochemicalseparation processes.
 93. The method for quantitatively separatingparticles with a multistage electromagnetic separator of claim 45,including the step of selecting homogeneous subpopulations of magneticparticles for targeted drug delivery.
 94. The method for quantitativelyseparating particles with a multistage electromagnetic separator ofclaim 45, including the step of classifying separation of magneticparticle according to magnetophoretic mobility and hence volumetricdifferential susceptibility.
 95. A method for quantitatively separatingparticles with a multistage electromagnetic separator for separatingparticles suspended in a fluid according to their magnetophoreticmobility, comprising a base unit including means for controlling, meansfor supporting, and means for providing electrical power to a platerotation apparatus, said plate rotation apparatus including atranslating electromagnet, an electromagnetic holding magnet, apermanent holding magnet, and a plate assembly including an upper platecontaining at least one collection cavity in selected fluidcommunication with and cooperatively engaging a second plate includingat least one sample container holding said particles in said fluiddisposed therein, said first plate moving with respect to said secondplate, means for moving said first plate with respect to said secondplate, means for aligning said first plate with respect to said secondplate, and means for controlling the movement of said first plate orsaid second plate, said translating electromagnet, and said holdingmagnet, comprising the steps of: filling said at least one samplecontainer through a sample port in said upper plate with a solutioncontaining at least some magnetic particles; rotating said upper plateto a full step position aligning said at least one collection cavity ofsaid upper plate with said at least one sample container of said lowerplate; activating said translation magnet energizing and moving saidparticles toward said lower plate and said upper plate interface;de-energizing said translating magnet; energizing said holdingelectromagnet; coupling holding electromagnetic field with a magneticfield of said permanent holding magnet; de-energizing said holdingelectromagnet; rotating said upper plate capturing a selected fractionof said particles in a selected collection cavity.
 96. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 95, wherein a sample container and its translatingmagnet serves an array of collection cavities.
 97. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 95, including the step of labeling said particleswith decreasing numbers of paramagnetic beads.
 98. The method forquantitatively separating particles with a multistage electromagneticseparator of claim 95, including the step of separating saidparamagnetic beads quantitatively on the basis of the extent of labelingby using magnetic fields of increasing strength.
 99. A method forquantitatively separating particles with a multistage electromagneticseparator for separating particles suspended in a fluid according totheir magnetophoretic mobility, comprising a base unit including meansfor controlling, means for supporting, and means for providingelectrical power to a plate rotation apparatus, said plate rotationapparatus including a translating electromagnet, an electromagneticholding magnet, a permanent holding magnet, and a plate assemblyincluding an upper plate containing at least one collection cavity inselected fluid communication with and cooperatively engaging a secondplate including at least one sample container holding said particles insaid fluid disposed therein, said first plate moving with respect tosaid second plate, means for moving said first plate with respect tosaid second plate, means for aligning said first plate with respect tosaid second plate, and means for controlling the movement of said secondplate with respect to said second plate, said translating electromagnet,and said holding magnet, comprising the steps of: half stepping rotationof said upper plate; filling said at least one collection cavity and atleast one sample container with fluid samples; rotating and aligningsaid at least one upper collection cavity into position above said atleast one sample container; energizing said translating electromagnet toa programmed current level creating a magnetic field within said atleast one sample container, subjecting said particles thereto, andattracting at least one particle with a specified mobility range;de-energizing said translational magnet; energizing said holdingelectromagnet to a programmed current level pulling said at least oneparticle within a specified mobility range into said at least onecollection cavity; de-energizing said holding electromagnet; holdingsaid at least one particle within a specified mobility range in positionwithin said collection cavity with said holding magnet; and rotatingsaid upper plate thereby capturing said at least one particle within aspecified mobility range.
 100. The method for quantitatively separatingparticles with a multistage electromagnetic separator for separatingparticles of claim 99, including the step of translating saidtranslating electromagnet from a bottom of said at least one samplecontainer to an interface of said upper plate and said lower plate. 101.The method for quantitatively separating particles with a multistageelectromagnetic separator for separating particles of claim 99,including the step of translating said translating electromagnet from aselected position along said at least one sample container to anotherselected position along said at least one sample container.
 102. Themethod for quantitatively separating particles with a multistageelectromagnetic separator for separating particles of claim 99,including the step of moving said at least one sample container withinan electric field produced by said translating electromagnet.
 103. Themethod for quantitatively separating particles with a multistageelectromagnetic separator for separating particles of claim 99,including the step of sequentially energizing a at least two translatingmagnets positioned from a bottom of said sample container to a top ofsaid sample container creating a magnetic field of different currentlevels.
 104. The method for quantitatively separating particles with amultistage electromagnetic separator for separating particles of claim99, including the step of sequentially energizing a at least twotranslating magnets positioned from a bottom of said sample container toa top of said sample container creating a magnetic field of differentcurrent levels.
 105. The method for quantitatively separating particleswith a multistage electromagnetic separator for separating particles ofclaim 99 wherein said holding magnet is selected from a group ofpermanent magnets comprising different sizes and materials.
 106. Themethod for quantitatively separating particles with a multistageelectromagnetic separator for separating particles of claim 99 includingthe step of suspending said particles in a solution of water,polyethylene glycol, alcohol, and combinations thereof.
 107. The methodfor quantitatively separating particles with a multistageelectromagnetic separator for separating particles of claim 99,including the step of using an index system to rotate said upper plate.108. The method of claim 1, including the step of: collecting saidparticles in a band by moving said magnet having a gap, from a bottom ofsaid at least one sample container to a selected different position;forming said electric field by translating said electromagnet upwardcarrying said migrating particles in the path of the gap; switching offsaid electromagnet upon said migrating particles reaching apredetermined level forming a band of migrating particles; activating aholding magnet positioned above said cavity causing said migratingparticles in said band within said sample container to migrate upward ata velocity proportional to their volumetric magnetic susceptibility;collecting said migrating particles in said collection cavity of saidupper plate; rotating said upper plate and said collection cavitycontaining said migrating particles sealing said collection cavity; andaligning a second collection cavity in fluid communication with saidsample container and repeating said process to quantitatively andqualitatively separate said particles contained in said sample containerin accordance with their magnetic susceptibility.